Expression and Functional Analysis of the Metallothionein and Metal-Responsive Transcription Factor 1 in Phascolosoma esculenta under Zn Stress

Metallothioneins (MTs) are non-enzymatic metal-binding proteins widely found in animals, plants, and microorganisms and are regulated by metal-responsive transcription factor 1 (MTF1). MT and MTF1 play crucial roles in detoxification, antioxidation, and anti-apoptosis. Therefore, they are key factors allowing organisms to endure the toxicity of heavy metal pollution. Phascolosoma esculenta is a marine invertebrate that inhabits intertidal zones and has a high tolerance to heavy metal stress. In this study, we cloned and identified MT and MTF1 genes from P. esculenta (designated as PeMT and PeMTF1). PeMT and PeMTF1 were widely expressed in all tissues and highly expressed in the intestine. When exposed to 16.8, 33.6, and 84 mg/L of zinc ions, the expression levels of PeMT and PeMTF1 in the intestine increased first and then decreased, peaking at 12 and 6 h, respectively, indicating that both PeMT and PeMTF1 rapidly responded to Zn stress. The recombinant pGEX-6p-1-MT protein enhanced the Zn tolerance of Escherichia coli and showed a dose-dependent ABTS free radical scavenging ability. After RNA interference (RNAi) with PeMT and 24 h of Zn stress, the oxidative stress indices (MDA content, SOD activity, and GSH content) and the apoptosis indices (Caspase 3, Caspase 8, and Caspase 9 activities) were significantly increased, implying that PeMT plays an important role in Zn detoxification, antioxidation, and anti-apoptosis. Moreover, the expression level of PeMT in the intestine was significantly decreased after RNAi with PeMTF1 and 24 h of Zn stress, which preliminarily proved that PeMTF1 has a regulatory effect on PeMT. Our data suggest that PeMT and PeMTF1 play important roles in the resistance of P. esculenta to Zn stress and are the key factors allowing P. esculenta to endure the toxicity of Zn.


Introduction
Heavy metal pollution is a global environmental problem [1].Human activities have increased the concentrations of heavy metals in water [2,3], which then accumulate in aquatic organisms through respiration, feeding, and skin penetration [4].Among heavy metals, zinc (Zn) is an essential trace element for organisms that participates in DNA replication, transcription, cell proliferation, and a variety of metabolic processes [5].However, excessive levels of Zn are harmful to aquatic organisms.Zn affects the stability of the lipid bilayer, thereby destroying the structure of enzymes and affecting the active transportation of cell membranes [6].In addition, it inhibits the absorption of other trace elements, resulting in anemia and enzyme inactivation [7].Moreover, studies have shown that excessive Zn causes oxidative stress, resulting in an imbalance of reactive oxygen species [8], and 2 of 16 influences apoptosis by acting on several molecular regulators of programmed cell death, including caspases and proteins from the Bcl and Bax families [9].
Metallothioneins (MTs) are non-enzymatic metal-binding proteins widely found in animals, plants, and microorganisms.They are the first line allowing organisms to endure the toxicity of heavy metals [10,11] and have the characteristics of low molecular weight and high cysteine (Cys) content [12].Their Cys residues can strongly chelate heavy metals and aid in detoxification [13,14], and the sulfhydryl group exists in a reduced state, allowing them to react with free radicals and act as antioxidants [15].Additionally, MTs play important roles in anti-apoptosis; high expression levels of MTs can enhance the anti-apoptosis ability of organisms, whereas low expression levels of MTs may promote apoptosis [16].The expression of MTs is regulated by metal-responsive transcription factor 1 (MTF1).As a zinc-induced transcription factor, MTF1 is activated by Zn 2+ and regulates MT by binding to metal-responsive elements (MREs) in the promoter of MT [17].MT and MTF1 play important roles in the regulation of Zn homeostasis, which protects the body from external stimuli and helps maintain normal physiological functions.Studies have shown that vertebrate MT and MTF1 homologs are highly conserved [18], whereas invertebrate MT and MTF1 homologs have low homology, and their functions require further exploration [19].
Phascolosoma esculenta is a valuable marine invertebrate belonging to the phylum Sipuncula [20].It inhabits intertidal zones and feeds on organic debris and benthic diatoms [21], which makes it vulnerable to heavy metal pollution.Previous studies have shown that P. esculenta has a high tolerance to heavy metal stress [22]; however, there is still a lack of research on the adaptation mechanism of P. esculenta in response to heavy metals, and whether PeMT and PeMTF1 are involved in the resistance to heavy metals has not been reported.
Therefore, in this study, we cloned and identified the PeMT and PeMTF1 genes and detected the tissue-specific expression of PeMT and PeMTF1 mRNA, and their temporal expression profiles under Zn stress.We then assessed the Zn tolerance of E. coli overexpressing pGEX-6p-1-MT and the in vitro antioxidant activity of the recombinant pGEX-6p-1-MT protein.Additionally, RNA interference (RNAi) and Zn stress were performed at the individual level, and changes in intestinal oxidative stress and apoptosis indices after interference with PeMT, as well as changes in intestinal PeMT mRNA expression after interference with PeMTF1, were detected.This study fills the gap in functional research on MT and MTF1 in Sipuncula and provides basic data for studying the molecular toxicology of P. esculenta.

Tissue-Specific Expression of PeMT and PeMTF1 mRNA
The relative expression levels of PeMT and PeMTF1 mRNA in the coelom fluid, intestine, constrictor muscle, nephridium, and body wall were detected using RT-PCR.The results showed that PeMT and PeMTF1 mRNA were widely expressed in all tissues.PeMT mRNA had the highest expression level in the intestine, followed by the coelom fluid, nephridium, constrictor muscle, and body wall (Figure 3A,B), whereas PeMTF1 mRNA showed the higher expression levels in the coelom fluid, constrictor muscle, and intestine than in the body wall and nephridium (Figure 3C,D).
than 50%, and green regions represent lower similarity.(E) Phylogenetic analysis of MTF1 homologous proteins.P. esculenta is shown in bold font, and PeMTF1 belongs to the invertebrate branch.

Tissue-Specific Expression of PeMT and PeMTF1 mRNA
The relative expression levels of PeMT and PeMTF1 mRNA in the coelom fluid, intestine, constrictor muscle, nephridium, and body wall were detected using RT-PCR.The results showed that PeMT and PeMTF1 mRNA were widely expressed in all tissues.PeMT mRNA had the highest expression level in the intestine, followed by the coelom fluid, nephridium, constrictor muscle, and body wall (Figure 3A,B), whereas PeMTF1 mRNA showed the higher expression levels in the coelom fluid, constrictor muscle, and intestine than in the body wall and nephridium (Figure 3C,D).

Expression Changes of PeMT and PeMTF1 mRNA in the Intestine under Zn Stress
The temporal expression profiles of PeMT and PeMTF1 mRNA in the intestine of P. esculenta, following exposure to different concentrations of Zn, are shown in Figure 4.No significant differences were observed in the control group (p > 0.05).However, they were upregulated first and then downregulated under Zn stress and reached the maximum at 12 and 6 h, respectively.When stressed for 24 and 48 h, the expression level of PeMT mRNA in the 84 mg/L group was significantly higher than that in the other groups (p < 0.05).After 96 h of Zn stress, the expression level of PeMT mRNA in the Zn stress groups was significantly lower than that in the control group (p < 0.05).Additionally, the expression level of PeMTF1 mRNA in the Zn stress groups was higher than that in the control group at all time points (except at 96 h of exposure to 84 mg/L Zn 2+ ).

Expression Changes of PeMT and PeMTF1 mRNA in the Intestine under Zn Stress
The temporal expression profiles of PeMT and PeMTF1 mRNA in the intestine of P. esculenta, following exposure to different concentrations of Zn, are shown in Figure 4.No significant differences were observed in the control group (p > 0.05).However, they were upregulated first and then downregulated under Zn stress and reached the maximum at 12 and 6 h, respectively.When stressed for 24 and 48 h, the expression level of PeMT mRNA in the 84 mg/L group was significantly higher than that in the other groups (p < 0.05).After 96 h of Zn stress, the expression level of PeMT mRNA in the Zn stress groups was significantly lower than that in the control group (p < 0.05).Additionally, the expression level of PeMTF1 mRNA in the Zn stress groups was higher than that in the control group at all time points (except at 96 h of exposure to 84 mg/L Zn 2+ ).

Prokaryotic Expression and Protein Purification of pGEX-6p-1-MT
The recombinant pGEX-6p-1-MT protein was obtained using a prokaryotic expression system.SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) analysis showed that it was expressed in the supernatant and precipitate of broken E. coli cells.Then, the supernatant was purified, and a single band with a molecular weight of 36.7 kDa was obtained, which was consistent with the theoretical value predicted by the Expasy ProtParam tool (Figure 5).The concentration of the purified pGEX-6p-1-MT protein was 1.44 mg/mL.

Zn Tolerance of the Recombinant E. coli
As shown in Figure 6, the growth rates of the bacteria that transformed pGEX-6p-1-MT and pGEX-6p-1 were almost the same in the control group, whereas they were inhibited by 0.3 mM Zn stress.The OD600 value of the recombinant strain was significantly

Prokaryotic Expression and Protein Purification of pGEX-6p-1-MT
The recombinant pGEX-6p-1-MT protein was obtained using a prokaryotic expression system.SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) analysis showed that it was expressed in the supernatant and precipitate of broken E. coli cells.Then, the supernatant was purified, and a single band with a molecular weight of 36.7 kDa was obtained, which was consistent with the theoretical value predicted by the Expasy ProtParam tool (Figure 5).The concentration of the purified pGEX-6p-1-MT protein was 1.44 mg/mL.

Prokaryotic Expression and Protein Purification of pGEX-6p-1-MT
The recombinant pGEX-6p-1-MT protein was obtained using a prokaryotic expression system.SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) analysis showed that it was expressed in the supernatant and precipitate of broken E. coli cells.Then, the supernatant was purified, and a single band with a molecular weight of 36.7 kDa was obtained, which was consistent with the theoretical value predicted by the Expasy ProtParam tool (Figure 5).The concentration of the purified pGEX-6p-1-MT protein was 1.44 mg/mL.

Zn Tolerance of the Recombinant E. coli
As shown in Figure 6, the growth rates of the bacteria that transformed pGEX-6p-1-MT and pGEX-6p-1 were almost the same in the control group, whereas they were inhibited by 0.3 mM Zn stress.The OD600 value of the recombinant strain was significantly

Zn Tolerance of the Recombinant E. coli
As shown in Figure 6, the growth rates of the bacteria that transformed pGEX-6p-1-MT and pGEX-6p-1 were almost the same in the control group, whereas they were inhibited by 0.3 mM Zn stress.The OD 600 value of the recombinant strain was significantly higher than that of the control strain after 2 h of Zn stress (p < 0.05) and reached an extremely significant level after 3 h of Zn stress (p < 0.01).
Int. J. Mol.Sci.2024, 25, x FOR PEER REVIEW 7 of 17 higher than that of the control strain after 2 h of Zn stress (p < 0.05) and reached an extremely significant level after 3 h of Zn stress (p < 0.01).

Changes in Intestinal Oxidative Stress and Apoptosis Indices after RNAi with PeMT and Zn Stress
Compared to the siNC group, the expression level of intestinal PeMT mRNA in the siMT group was significantly decreased by 46.89% after RNAi and 24 h of Zn stress (p <

Changes in Intestinal Oxidative Stress and Apoptosis Indices after RNAi with PeMT and Zn Stress
Compared to the siNC group, the expression level of intestinal PeMT mRNA in the siMT group was significantly decreased by 46.89% after RNAi and 24 h of Zn stress (p <

Changes in Intestinal Oxidative Stress and Apoptosis Indices after RNAi with PeMT and Zn Stress
Compared to the siNC group, the expression level of intestinal PeMT mRNA in the siMT group was significantly decreased by 46.89% after RNAi and 24 h of Zn stress (p < 0.05) (Figure 8).At the same time, the malondialdehyde (MDA) content, superoxide dismutase (SOD) activity, and glutathione (GSH) content were significantly increased to 0.05) (Figure 8).At the same time, the malondialdehyde (MDA) content, superoxide dismutase (SOD) activity, and glutathione (GSH) content were significantly increased to 169%, 137.76%, and 139.62% of the siNC group, respectively (p < 0.05) (Figure 9A-C).Moreover, the Caspase 3, Caspase 8, and Caspase 9 activities were significantly increased to 134.14%, 123.64%, and 128.13% of the siNC group, respectively (p < 0.05) (Figure 9D-F).

Changes in Intestinal PeMT mRNA Expression Level after RNAi with PeMTF1 and Zn Stress
Compared to the siNC group, the expression level of intestinal PeMTF1 mRNA in the siMTF1 group was significantly decreased by 42.69% (p < 0.05) (Figure 10A), and the expression level of intestinal PeMT mRNA decreased by 53.95% (p < 0.01) (Figure 10B).

Changes in Intestinal PeMT mRNA Expression Level after RNAi with PeMTF1 and Zn Stress
Compared to the siNC group, the expression level of intestinal PeMTF1 mRNA in the siMTF1 group was significantly decreased by 42.69% (p < 0.05) (Figure 10A), and the expression level of intestinal PeMT mRNA decreased by 53.95% (p < 0.01) (Figure 10B).

Changes in Intestinal PeMT mRNA Expression Level after RNAi with PeMTF1 and Zn Stress
Compared to the siNC group, the expression level of intestinal PeMTF1 mRNA in the siMTF1 group was significantly decreased by 42.69% (p < 0.05) (Figure 10A), and the expression level of intestinal PeMT mRNA decreased by 53.95% (p < 0.01) (Figure 10B).after RNAi with PeMTF1 and 24 h of Zn stress.β-actin gene was used as an internal reference, and all data are presented as the mean ± SD (n = 4), "*": p < 0.05, "**": p < 0.01.

Sequence Features and Protein Structure of PeMT and PeMTF1
The full-length PeMT cDNA was cloned from P. esculenta.The "CKCSKCCPCA" was in line with the conserved motif "CKCXXXCXCX" of invertebrate MTs, and the "CNCG-DACSCKEGCKCK" in the C-terminus was similar to the conserved motif "CXCXXXCT-GXXXCXCXXXCXCK" of Mollusca MTs [23].Moreover, the conserved polyadenylation signal "AATAAA" was observed in the 3 ′ -UTR, which is crucial for mRNA localization and translation [24] and has been found in the MTs of Mollusca [25], Crustacea [26], and Pisces [27].PeMT showed low similarity to other homologs, and similar results have been found in invertebrate MTs of Hermetia illucens [28], Oxya chinensis [29], and Apostichopus japonicus [30].PeMT contains 28 Cys and 12 Lys residues.These Cys residues exist in the motifs of Cys-Cys, Cys-X-Cys, Cys-X-X-Cys, and Cys-X-X-X-Cys, which show great affinity for binding heavy metals such as Zn, Cu, and Cd, and participate in divalent detoxification and maintain the stability of metal binding clusters together with Lys [25,31,32].Studies have shown that the tertiary structure of vertebrate MTs is conserved, consisting of an α domain and a β domain, which is dumbbell-shaped [33].However, it varies among invertebrate MTs.For example, Charybdis japonica and Portunus trituberculatus [31,32] MTs consist of two β domains.The Crassostrea gigas MT1 consists of an α domain and a β domain, and the C. gigas MT2 consists of an α domain and two β domains [34].Moreover, the Drosophila melanogaster MT has only one domain [35].In this study, PeMT was also composed of one domain, further proving that MTs convergently evolved.
Additionally, the full-length PeMTF1 cDNA was cloned.PeMTF1 was predicted to be located in the cytoplasm and nucleus.Previous studies have shown that MTF1 is located in the cytoplasm and nucleus under normal conditions.However, when cells are stimulated by heavy metals, hypoxia, and other external stimuli.MTF1 may translocate to the nucleus and bind to serval target genes [17].Moreover, sequence analysis showed that the N-terminus of PeMTF1 has six conserved C 2 H 2 -type zinc finger domains that have high homology with other homologs.The zinc finger domains play important roles in metal regulation and DNA binding [36], and their high conservation indicates that they may have conserved functions.However, in other regions, PeMTF1 showed low similarity to other homologs and lacked transactivation domains.In a study by Ren et al. [37], MTF1 homologs of Cerapachys biroi, Hymenoptera, and Oxya chinesis showed similar characteristics.In addition, Ferencz et al. [38] found that the splicer MTF1-1.1a of Cyprinus carpio has six zinc finger domains and a truncated acidic domain but lacks proline and serine/threonine domains.Based on these results, we identified PeMTF1 as a new member of the MTF1 superfamily.

Tissue-Specific Expression of PeMT and PeMTF1 and Their Expression Changes under Zn Stress
Extensive studies have reported that MT is widely expressed in all vertebrate and invertebrate tissues [39], and tissues with high expression levels of MT were determined to be different among species.For example, Meretrix meretrix [40], Haliotis discus hannai [41], and Litopenaeus vannamei [42] showed the highest MT expression levels in the liver, while MTs of liverless species such as A. japonicus [30], H. illucens [28], and O. chinensis [29] were highly expressed in the intestine.Consistent with these findings, PeMT was widely expressed in the tested tissues, with the intestine showing the highest expression.The intestine is one of the main organs involved in nutrient absorption and immune defense [43,44], and high expression of PeMT indicates that PeMT plays an important role in the intestine of P. esculenta.Therefore, we further analyzed the temporal expression profile of intestinal PeMT under four concentrations (0, 16.8, 33.6, and 84 mg/L) of Zn 2+ .The expression pattern of PeMT showed an inverted "U" shape as the stress time increased, which is consistent with previous studies [45].For instance, when exposed to Cd and Zn, the expression level of MT in the gills of Septifer virgatus increased significantly and reached a maximum after 5 days [46].In C. japonica, the expression level of MT significantly increased within 12 h of Pb stress and then slowly decreased [47].Additionally, A. japonicus MT displayed the same trend under Cd and Zn stress [30].The increase in MTs in the early stages indicates that MTs rapidly respond to heavy metal stress [46], whereas the decrease in the later stages may be related to peroxidation stress.These findings confirm our speculation that PeMT is a functional gene involved in Zn detoxification in P. esculenta and is expected to be a biomarker for monitoring Zn pollution.
PeMTF1 was also expressed in the tested tissues, and its expression levels in the coelom fluid, constrictor muscle, and intestine were higher than those in the body wall and nephridium.Under Zn stress, it showed the same trend as PeMT and reached the maximum at 6 h.In C. carpio, the expression of MTF1-1.1a was first upregulated and then downregulated under Cd stress, reaching a maximum at 48 h [38].In the liver of Pelteobagrus fulvidraco, the expression levels of MTF1 and MT significantly increased within 48 h of Zn stress [17].In C. gigas, the expression levels of MTF1 and MT were induced under 0.1 ppm Cd, and the response of MTF1 was faster than that of MT [48].These findings indicate that MTF1 plays an important role in the response to oxidative stress induced by heavy metals, and its response is faster than that of MT, which may be involved in the regulation of MT.

Detoxification, Antioxidant, and Anti-Apoptosis Functions of PeMT
MTs play an important role in heavy metal detoxification and increase metal tolerance in organisms [49].Wang et al. [50] inserted Sinopotamon MT into the pGEX-4t-1 vector and exposed it to varying concentrations of Cd, Cu, and Zn.It was found that the recombinant strain showed stronger metal tolerance than the control group.Yang et al. [40] constructed the recombinant pET32a-MnMT and found that it could bind to Cu 2+ and Cd 2+ , making the recombinant strain more resistant to toxic heavy metals.Li et al. [51] constructed the recombinant pET28a-NtMT2F and found that the growth rate of the recombinant strain was faster than that of the control group when exposed to Cd.In the present study, we induced the expression of PeMT in E. coli and exposed the cells to Zn 2+ .The results showed that Zn 2+ inhibited the growth of E. coli, but the growth of the recombinant strain was better than that of the control.Based on these results, we believe that PeMT exerts detoxifying effects and enhances Zn tolerance in E. coli.
The sulfhydryl group of MTs exists in a reduced state and can react with free radicals to exert an antioxidant function [52,53].Recombinant human MT-III has been reported to reduce the oxidative damage caused by Cu and Cd stress [54].Purified Channa punctate MT has been shown to have in vitro activities; it plays an important role in scavenging superoxide radicals (O 2− ), ABTS radicals, and DPPH radicals and can protect the body from oxidative damage induced by Fe-NTA [55].In this study, the recombinant pGEX-6p-1-MT protein was used to conduct an in vitro experiment.The results showed that PeMT exhibited ABTS radical scavenging ability in a dose-dependent manner.These findings indicated that PeMT has antioxidant functions and plays an important role in resisting Zn-induced oxidative stress.
To further investigate the functions of PeMT, we performed RNAi at the individual level.After RNAi with PeMT and 24 h of Zn stress, the expression level of PeMT was significantly decreased by 46.89%, and the oxidative indices (MDA, SOD, and GSH) and apoptosis indices (Caspase 3, Caspase 8, and Caspase 9) were significantly increased.MDA is the product of lipid peroxidation, and its content is related to the degree of oxidative damage [56].SOD is the first barrier of organisms' antioxidant defense, and it can disproportionate the superoxide anion (O 2− ) to H 2 O 2 and O 2 [57].GSH acts as the substrate of GSH-Px and is involved in the removal of H 2 O 2 [58].Additionally, when oxidative stress exceeds physiological tolerance, Caspase 8 and Caspase 9 can bind to apoptotic signals, thus activating Caspase 3 and causing apoptosis [59].In this study, the oxidative stress and apoptosis indices of the siMT group were significantly increased, indicating that the degree of oxidative damage and apoptosis was more severe in the siMT group, which further proved that PeMT plays an important role in antioxidation and anti-apoptosis.

Regulation Effect of PeMTF1 on PeMT
To explore the regulatory effects of PeMTF1 on PeMT, we performed RNAi experiments at the individual level.After RNAi with PeMTF1 and 24 h of Zn stress, the expression level of PeMT significantly decreased (p < 0.05).Similarly, Meng et al. [60] found that the expression levels of CgMT1 and CgMT4 in the gills of C. gigas were significantly decreased after RNAi with CgMTF1 and 48 h of Zn stress.Ren et al. [37] found that the expression level of OcMT in O. chinensis significantly decreased after RNAi with OcMTF1.Troadec et al. [61] knocked down MTF1 in NIH 3T3 cells and found that the expression level of MT was significantly decreased.Moreover, Atanesyan et al. [62] found that the MTnE of Drosophila was almost unexpressed after RNAi with MTF1 and was highly expressed after the overexpression of MTF1.These findings suggest that PeMTF1 has a conserved function and participates in the regulation of PeMT.PeMT and PeMTF1 are key factors allowing P. esculenta to endure the toxicity of heavy metal pollution.

Animals
P. esculenta individuals (3.9 ± 0.5) g were collected from Xizhou of Ningbo, Zhejiang, China.They were then kept in filtered seawater at a temperature of 20.5 ± 0.5 • C, salinity of 25.5 ± 0.5‰, and pH of 7.6 ± 0.2 for four days.

Treatments and Sampling
Preliminary experiments showed that the 96 h median lethal concentration (LC 50 ) of Zn 2+ for P. esculenta was 167.909 mg/L.Based on this value, four Zn 2+ concentrations (0, 16.8, 33.6, and 84 mg/L) were set for 96 h of stress.Three tanks were used for each concentration, and 30 individuals were placed in each tank.The seawater was changed daily, and the Zn concentrations were kept constant.
Samples of the coelom fluid, intestine, constrictor muscle, nephridium, and body wall were collected from six individuals in the control group at 0 h, and the intestines of six individuals at each concentration were sampled after 6, 12, 24, 48, and 96 h of Zn stress.At each time point, two individuals were randomly selected from each tank, they were then sampled and mixed into one sample.The coelom fluid was extracted with a 1 mL syringe.Then, P. esculenta individuals were placed on the ice, and the intestines, constrictor muscles, nephridia, and body walls were collected and washed with distilled water.Finally, the samples were immediately frozen in liquid nitrogen and then stored at −80 • C. All experimental procedures were approved by the Animal Care and Use Committee of Ningbo University (Ningbo, China).

RNA Extraction and cDNA Synthesis
The RNA-Solv Reagent (Omega, GA, USA) was used to extract total RNA from the tissues of P. esculenta.Some of the obtained RNA was reverse transcribed by PrimeScript ® RT Kit (Takara, Dalian, China) for intermediate fragment cloning and tissue expression analysis, and the remaining was reverse transcribed by SMARTer RACE 5 ′ /3 ′ reagent (Takara, Dalian, China) for 5 ′ and 3 ′ RACE (rapid amplification of cDNA ends).

Full-Length cDNA Cloning of PeMT and PeMTF1
We obtained the ORFs of PeMT and PeMTF1 from the transcriptome data of P. esculenta (GenBank Accession No. OL757513) and designed specific primers using Primer Premier 5.0 for verification.Based on the verification results, specific primers for 5 ′ and 3 ′ RACE were designed, and full-length cloning was carried out.The primers used are listed in Table S1.
The PCR procedures involved the following steps: 94 The multiple sequence alignment and phylogenetic tree analysis of the MT and MTF1 homologs were performed by Vector NTI 11.5 and MEGA 5.1 software.The species and their GenBank Accession numbers used in the multiple sequence alignment and phylogenetic tree analysis are listed in Tables S2 and S3.RT-qPCR was conducted to study the temporal expression profiles of intestinal PeMT and PeMTF1 mRNA under Zn stress.The PCR procedures were as follows: 95 • C 5 min; 40 cycles of 95 • C 30 s, 55 • C 30 s, and 72 • C 30 s; 72 • C 5 min.Finally, the relative expression level of PeMT and PeMTF1 mRNA was calculated by the 2 −∆∆CT method.All data are presented as mean ± SD (n = 3).The primers used are listed in Table S1.

Expression and Purification of the Recombinant pGEX-6p-1-MT
Primers with restriction sites (BamHI and XhoI) were used to amplify the ORF of PeMT.The PCR product and pGEX-6p-1 vector were double digested with BamHI and XhoI restriction enzymes (Thermo Fisher Scientific, Waltham, MA, USA) and then ligated by T4 ligase (Takara, Dalian, China).The recombinant plasmid was transformed into Trans5α (TransGen Biotech, Beijing, China) and cultured overnight on a solid LB medium with 100 µg/mL of Ampicillin.The following day, positive clones were screened, and plasmids were extracted by the Plasmid Extraction Mini Kit (Solarbio, Beijing, China).
After sequencing, the correct recombinant plasmid was transformed into Transetta (DE3) (TransGen Biotech, Beijing, China) and cultured in the liquid LB medium with 100 µg/mL Ampicillin at 37 • C and 180 rpm.When the OD 600 reached a density of 0.5-0.7, 1 mM of IPTG was added to induce the expression of the recombinant protein.Based on the SDS-PAGE analysis, the supernatant was purified by the GST-tag Protein Purification Kit (Beyotime, Shanghai, China).

Zn Tolerance of the Recombinant E. coli
The recombinant E. coli strains that transformed pGEX-6p-1-MT and pGEX-6p-1 were cultured in liquid LB medium with 100 µg/mL Ampicillin until the OD 600 reached 0.4, and then 1 mM of IPTG was added.They were then divided into control and 0.3 mM Zn stress groups based on our pre-experiment (Figure S1), and their OD 600 was measured in the following 8 h.The results are presented as mean ± SD (n = 3).

Antioxidant Capacity of the Recombinant pGEX-6p-1-MT
The pGEX-6p-1 protein was purified as described previously.We determined the concentrations of pGEX-6p-1 and pGEX-6p-1-MT using the Bradford Protein Concentration Assay Kit (Beyotime, Shanghai, China) and prepared 1 mg/mL of GSH.Then, they were diluted to 0.12, 0.18, 0.24, and 0.3 mg/mL, and the ABTS free radical scavenging ability was determined by the Total Antioxidant Capacity Assay Kit (Beyotime, Shanghai, China).

Detection of Intestinal Oxidative Stress and Apoptosis Indices after RNAi with PeMT and Zn Stress
Interference primers were designed and synthesized by Shanghai GenePharma Technology Co., Ltd.(GenePharma, Shanghai, China) (Table S1).Eight P. esculenta individuals (3.9 ± 0.5 g) were divided into siNC and siMT groups.Lipo6000 TM (Beyotime, Shanghai, China) and siRNA were diluted with 1 × PBS at a volume ratio of 17:3 and mixed at a volume ratio of 1:1.After mixing, the mixture was stored at room temperature for 5 min and injected at a dose of 1 µg/g.Then, P. esculenta individuals were exposed to 33.6 mg/L Zn for 24 h.The intestines were then collected and stored at −80 • C.
The interference efficiency of PeMT mRNA was determined by RT-qPCR using the method described above.Intestines were accurately weighed and mixed with extract solution in the ratio of weight (g)/volume (mL) = 1:10.The mixture was then homogenized in an ice bath and centrifuged at 10,000× g for 8 min at 4 • C. The oxidative stress indices (MDA, SOD, and GSH) and apoptosis indices (Caspase 3, Caspase 8, and Caspase 9) were measured by assay kits according to the instructions (Solarbio, Beijing, China).

Detection of Intestinal PeMT mRNA Expression Level after RNAi with PeMTF1 and Zn Stress
The RNAi method for PeMTF1 was similar to that for PeMT.After sampling, the interference efficiency of PeMTF1 mRNA and changes in the expression of PeMT mRNA were detected by RT-qPCR, as described above.
4.12.Statistical Analysis SPSS 26.0, Excel 2016, and GraphPad Prism 8.0 were used for statistical analysis, and significance was tested using one-way ANOVA followed by Duncan's test and independent samples t-test.Different letters simultaneously indicate a significant difference among the groups (p < 0.05).

Conclusions
PeMT and PeMTF1 were cloned and identified from P. esculenta.Their expression patterns indicate that they respond rapidly to Zn stress.Prokaryotic expression, biochemical detection, and RNAi experiments provided evidence for the functions of PeMT and PeMTF1, suggesting that PeMT plays an important role in Zn detoxification, antioxidation, and antiapoptosis, and its expression is regulated by PeMTF1, proving that PeMT and PeMTF1 are key factors allowing P. esculenta to endure the toxicity of Zn.This study made up for the gap in the functional research of MT and MTF1 in Sipuncula and accumulated basic data for studying the molecular toxicology of P. esculenta.

Figure 1 .
Figure 1.Full-length cloning and bioinformatic analysis of PeMT.(A) Completed cDNA and deduced amino acid sequence of PeMT.The red font is the start codon and the end codon.The red underline is a putative polyadenylation signal.The blue shadow is the characteristic sequence of invertebrate MTs.The yellow shadow is similar to the characteristic sequence of Mollusca MTs.* represents the termination codon.(B) N-terminus and C-terminus of PeMT.(C) Yellow parts show the conserved Cys residues, and gray spheres indicate Zn 2+ .(D) Multiple sequence alignment of PeMT.The similarities between PeMT and its homologs in A. plicatula, C. gigas, S. cumingii, C.edule, P. viridis, D. rerio, O. latipes, M. gallus, and H. sapiens were 37.1%, 36.8%,33.3%, 35.2%, 32.4%, 25.7%, 20.0%, 17.1%, and 16.2%, respectively.The same amino acid residues are shaded in yellow, blue regions indicate amino acid residues with a similarity greater than 50%, and green regions represent lower similarity.(E) Phylogenetic analysis of MT homologous proteins.P. esculenta is shown in bold font, and PeMT belongs to the invertebrate branch.

Figure 1 .
Figure 1.Full-length cloning and bioinformatic analysis of PeMT.(A) Completed cDNA and deduced amino acid sequence of PeMT.The red font is the start codon and the end codon.The red underline is a putative polyadenylation signal.The blue shadow is the characteristic sequence of invertebrate MTs.The yellow shadow is similar to the characteristic sequence of Mollusca MTs.* represents the termination codon.(B) N-terminus and C-terminus of PeMT.(C) Yellow parts show the conserved Cys residues, and gray spheres indicate Zn 2+ .(D) Multiple sequence alignment of PeMT.The similarities between PeMT and its homologs in A. plicatula, C. gigas, S. cumingii, C. edule, P. viridis, D. rerio, O. latipes, M. gallus, and H. sapiens were 37.1%, 36.8%,33.3%, 35.2%, 32.4%, 25.7%, 20.0%, 17.1%, and 16.2%, respectively.The same amino acid residues are shaded in yellow, blue regions indicate amino acid residues with a similarity greater than 50%, and green regions represent lower similarity.(E) Phylogenetic analysis of MT homologous proteins.P. esculenta is shown in bold font, and PeMT belongs to the invertebrate branch.
invertebrate and vertebrate branches.PeMTF1 clustered in the invertebrate branch and had the closest relationship with MTF1 from Strongylocentrotus purpuratus among the selected species (Figure2E).

Figure 2 .
Figure 2. Full-length cloning and bioinformatic analysis of PeMTF1.(A) Completed cDNA and deduced amino acid sequence of PeMTF1.The red shadow is the start codon and the end codon.The yellow shadows are the C2H2-type zinc finger domains.* represents the termination codon.(B) Nterminus and C-terminus of the protein sequence.(C) Yellow parts are the conserved C2H2-type zinc finger domains.(D) Multiple sequence alignment of PeMTF1.The similarities between PeMTF1 and its homologs in C. pelagica, G. gallus, H. sapiens, M. musculus, R. norvegicus, C. tigris, L. agilis,and D. rerio were 36.4%,37.0%, 34.4%, 37.0%, 38.7%, 39.9%, 36.5%, and 43.0%, respectively, and the similarities of the zinc finger domains were 84.0%, 84.0%, 84.6%, 84.6%, 84.6%, 84.6%, 84.6%, and 85.7%, respectively.The red boxes are six conserved C2H2-type zinc finger domains.The same amino acid residues are shaded in yellow, blue regions indicate amino acid residues with a similarity greater

Figure 2 .
Figure 2. Full-length cloning and bioinformatic analysis of PeMTF1.(A) Completed cDNA and deduced amino acid sequence of PeMTF1.The red shadow is the start codon and the end codon.The yellow shadows are the C 2 H 2 -type zinc finger domains.* represents the termination codon.(B) N-terminus and C-terminus of the protein sequence.(C) Yellow parts are the conserved C 2 H 2type zinc finger domains.(D) Multiple sequence alignment of PeMTF1.The similarities between PeMTF1 and its homologs in C. pelagica, G. gallus, H. sapiens, M. musculus, R. norvegicus, C. tigris,L.agilis, and D. rerio were 36.4%,37.0%, 34.4%, 37.0%, 38.7%, 39.9%, 36.5%, and 43.0%, respectively, and the similarities of the zinc finger domains were 84.0%, 84.0%, 84.6%, 84.6%, 84.6%, 84.6%, 84.6%, and 85.7%, respectively.The red boxes are six conserved C 2 H 2 -type zinc finger domains.The same amino acid residues are shaded in yellow, blue regions indicate amino acid residues with a similarity greater than 50%, and green regions represent lower similarity.(E) Phylogenetic analysis of MTF1 homologous proteins.P. esculenta is shown in bold font, and PeMTF1 belongs to the invertebrate branch.

Figure 3 .
Figure 3. Tissue-specific expression of PeMT and PeMTF1 mRNA.(A) RT-PCR detection of PeMT mRNA.(B) Gray value of RT-PCR analyzed using Image J. (C) RT-PCR detection of PeMTF1 mRNA.(D) Gray value of RT-PCR analyzed using Image J. β-actin gene was used as an internal reference, and all data are presented as the mean ± SD (n = 3).Different letters indicate significant differences among the tissues (p < 0.05).

Figure 3 .
Figure 3. Tissue-specific expression of PeMT and PeMTF1 mRNA.(A) RT-PCR detection of PeMT mRNA.(B) Gray value of RT-PCR analyzed using Image J. (C) RT-PCR detection of PeMTF1 mRNA.(D) Gray value of RT-PCR analyzed using Image J. β-actin gene was used as an internal reference, and all data are presented as the mean ± SD (n = 3).Different letters indicate significant differences among the tissues (p < 0.05).

Figure 4 .
Figure 4. Expression changes of (A) PeMT and (B) PeMTF1 mRNA in the intestine of P. esculenta under Zn stress.β-actin gene was used as an internal reference, and all data are presented as the mean ± SD (n = 3).Different letters indicate significant differences among the groups (p < 0.05).

Figure 5 .
Figure 5. Expression and purification of the recombinant pGEX-6p-1-MT.Line M: marker; Line 1 shows the cell lysate of pGEX-6p-1-MT without induction; Line 2 shows the cell lysate of pGEX-6p-1-MT induced by 1 mM IPTG (isopropyl-β-D-thiogalactoside); Line 3 shows the supernatant of cell lysate; Line 4 shows the precipitation of cell lysate; Lines 5-7 show the purified protein; black arrow: the expressed and purified protein.

Figure 4 .
Figure 4. Expression changes of (A) PeMT and (B) PeMTF1 mRNA in the intestine of P. esculenta under Zn stress.β-actin gene was used as an internal reference, and all data are presented as the mean ± SD (n = 3).Different letters indicate significant differences among the groups (p < 0.05).

Figure 4 .
Figure 4. Expression changes of (A) PeMT and (B) PeMTF1 mRNA in the intestine of P. esculenta under Zn stress.β-actin gene was used as an internal reference, and all data are presented as the mean ± SD (n = 3).Different letters indicate significant differences among the groups (p < 0.05).

Figure 5 .
Figure 5. Expression and purification of the recombinant pGEX-6p-1-MT.Line M: marker; Line 1 shows the cell lysate of pGEX-6p-1-MT without induction; Line 2 shows the cell lysate of pGEX-6p-1-MT induced by 1 mM IPTG (isopropyl-β-D-thiogalactoside); Line 3 shows the supernatant of cell lysate; Line 4 shows the precipitation of cell lysate; Lines 5-7 show the purified protein; black arrow: the expressed and purified protein.

Figure 5 .
Figure 5. Expression and purification of the recombinant pGEX-6p-1-MT.Line M: marker; Line 1 shows the cell lysate of pGEX-6p-1-MT without induction; Line 2 shows the cell lysate of pGEX-6p-1-MT induced by 1 mM IPTG (isopropyl-β-D-thiogalactoside); Line 3 shows the supernatant of cell lysate; Line 4 shows the precipitation of cell lysate; Lines 5-7 show the purified protein; black arrow: the expressed and purified protein.

Figure 7 .
Figure 7.The ABTS free radical scavenging ability of the recombinant PeMT.Different concentrations of pGEX-6p-1 and GSH were set as the control groups.All data are presented as the mean ± SD (n = 3); different letters indicate significant differences among the groups (p < 0.05).
Int. J. Mol.Sci.2024, 25, x FOR PEER REVIEW 7 of 17 higher than that of the control strain after 2 h of Zn stress (p < 0.05) and reached an extremely significant level after 3 h of Zn stress (p < 0.01).

Figure 7 .
Figure 7.The ABTS free radical scavenging ability of the recombinant PeMT.Different concentrations of pGEX-6p-1 and GSH were set as the control groups.All data are presented as the mean ± SD (n = 3); different letters indicate significant differences among the groups (p < 0.05).

Figure 7 .
Figure 7.The ABTS free radical scavenging ability of the recombinant PeMT.Different concentrations of pGEX-6p-1 and GSH were set as the control groups.All data are presented as the mean ± SD (n = 3); different letters indicate significant differences among the groups (p < 0.05).

Figure 8 .
Figure 8. Changes in the relative expression of PeMT mRNA after RNAi with PeMT and 24 h of Zn stress.β-actin gene was used as an internal reference, and all data are presented as the mean ± SD (n = 4), "*": p < 0.05.

Figure 8 .
Figure 8. Changes in the relative expression of PeMT mRNA after RNAi with PeMT and 24 h of Zn stress.β-actin gene was used as an internal reference, and all data are presented as the mean ± SD (n = 4), "*": p < 0.05.

Figure 8 .
Figure 8. Changes in the relative expression of PeMT mRNA after RNAi with PeMT and 24 h of Zn stress.β-actin gene was used as an internal reference, and all data are presented as the mean ± SD (n = 4), "*": p < 0.05.

17 Figure 10 .
Figure10.The expression changes of (A) PeMTF1 and (B) PeMT mRNA in the intestine of P. esculenta after RNAi with PeMTF1 and 24 h of Zn stress.β-actin gene was used as an internal reference, and all data are presented as the mean ± SD (n = 4), "*": p < 0.05, "**": p < 0.01.

Figure 10 .
Figure 10.The expression changes of (A) PeMTF1 and (B) PeMT mRNA in the intestine of P. esculenta

4. 6 .
Tissue-Specific Expression of PeMT and PeMTF1 mRNA and Its Expression Changes under Zn Stress Tissue-specific expression of PeMT and PeMTF1 mRNA in the coelom fluid, intestine, constrictor muscle, nephridium, and body wall was monitored by RT-PCR, with the β-actin serving as an internal reference.The PCR procedures were as follows: 94 • C 5 min; 30 cycles of 94 • C 15 s, 55 • C 15 s, and 72 • C 15 s; 72 • C 5 min.The PCR products were separated by 1% agarose gel electrophoresis, visualized by a gel image analysis system (FR, Shanghai, China), and then analyzed by Image J software.All data are presented as mean ± SD (n = 3).